Nature - USA (2020-09-24)

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nature research | reporting summary

October 2018

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size For CRISPR injected embryos in each experiment a group of at least 100 embryos were injected with the corresponding reagents in a plate.

After developing to the appropriate stage alive embryos without gross morphological defects were used for subsequent experiments. Fig 1b,

1c, 1f, 1g and 2b.

For expression analyses via ISH at least 10 embryos per condition were analyzed. Detail as follows:

For spaw, myl7, prrx1a, snail1b and twist1a expression embryos were hybridized in groups of 30. Fig 1c, 1f, 1g and 2b.

For amhc immunodetection groups of 10 embryos were analyzed Fig 1d

For prrx1 inmunodetection a group of 10 embryos of each condition was used Fig 1b

For a-tubulin detection groups of 10 embryos were analyzed Fig 1e

We did not performed a prehoc test to determine sample size. The sample size for the injection experiments was chosen to be 100 the

minimum to have enough surviving embryos to analyse. In most cases the sample size was larger than the minimum.

For in situ hybridizations 30 embryos per group were chosen since this is the size that can be handled properly following our SEP.

Immunodetections: we have chosen 10 to prevent antibody dilution due to the volume of the samples.

In the hybridization and immunodetection experiments we defined the experiment successful only if at least a 80% of the embryos showed

the same pattern.

Data exclusions After injection with CRISPR components embryos that were dead or had stopped their development were excluded

After immunodetection or in situ hybridization samples that were damaged, showed no expression or high background were excluded from

the analysis

Replication All replication attempts of the experiments were successful. Al experiments were repeated at least twice in different days using the same

experimental procedures. In the injection experiments there was always a set of embryos from the same clutch that was kept in similar

incubation conditions. Experiments were discarded when the control embryos showed malformations.

Duplicates were analysed for differences from the other replicates, we did nor detect any differences between replicates.

Randomization All animals were randomly selected for experiments.

Blinding Blinding was not possible since in most cases it was possible to identify which sample was being scored. To prevent biases, at least two

individuals checked the analysis of experiments. We defined a scoring system for heart position that was applied to every experiment. See

Rago et al 2019 Dev Cell for details

Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,

system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

n/a Involved in the study

Antibodies

Eukaryotic cell lines

Palaeontology

Animals and other organisms

Human research participants

Clinical data

Methods

n/a Involved in the study

ChIP-seq

Flow cytometry

MRI-based neuroimaging

Antibodies

Antibodies used Rabbit anti Prrx1 (for IF). Tanaka lab. Used at 1:200

Alexa Fluor 488 goat anti-rabbit. Invitrogen. A11008. Used at 1:500

Mouse anti-aetylated alpha-tubulin. Sigma. T6793. Used at 1:600

Alexa Fluor 568 goat anti-mouse Invitrogen. A11004. Used at 1:500

Chicken IgY anti- GFP. Aveslab (2BScientific), GFP-1020. Used at 1:500

Alexa Fluor 488 goat anti-chicken IgG (H+L). Life Technologies, A11039).Used at 1:500

DIG-AP Fab fragments: sheep polyclonal. Roche (11093274910) Used at 1:500

Validation Rabbit anti Prrx1 has been validated for zebrafish in Ocana et al. 2017, Nature.

Mouse anti-acetylated alpha-tubulin. Sigma T6793. Specificity is described in the Sigma web page and it was tested in our lab in

control zebrafish AB embryos.

Chicken IgY anti- GFP Aveslab (2BScientific), GFP-1020 specificity is described by the manufacturer and was tested in our lab by